PROJECT SUMMARY/ABSTRACT: Traumatic brain injury (TBI) leads to secondary neuropsychiatric complications that develop and persist years after injury and negatively affect health span. Mounting evidence indicates that neuroinflammatory processes advance after the initial head injury and worsen with time. The ongoing inflammatory processes after TBI are mediated by microglia and astrocytes. In fact, these glia are the primary inflammatory responders to the injury. Therefore, it is important to identify interventions that can be provided immediately after TBI to reduce glial- mediated inflammation and facilitate recovery. It is also critical that immediate interventions prevent the development of TBI-related long-term complications in behavior, cognition, and pathology. New data are provided to show that immediate intervention with methylene blue (MB), an antioxidant and anti-inflammatory agent, reduces neuroinflammation in mice after moderate and diffuse TBI, improves functional recovery, and limits the development of primed and reactive microglia 1 month after injury. Development of a primed microglial phenotype is relevant because it represents an increased state of inflammation. These primed glia are highly reactive to subsequent immune challenges, which trigger the development of neuropsychiatric complications. Overall, we show that there are both acute and long-term benefits of immediate MB intervention after TBI. MB is used clinically in sepsis, ischemia, and vasoplegic syndrome but it has not been used clinically for TBI. MB is safe, crosses the blood brain barrier, and is administered intravenously. Therefore, our goal is to determine the degree to which MB intervention shifts the activation profile of microglia and astrocytes and protects against secondary complications following TBI including cognitive decline, glia-mediated inflammation, and glial reactivity to immune challenge. To address this, three objectives are proposed using a midline fluid percussion injury model of TBI in mice. In Aim-1 we will ascertain if MB intervention after TBI promotes a neuroprotective ?repair? profile of microglia and astrocytes. We will use unique approaches to determine the effects of TBI and MB intervention specifically on microglia and astrocyte mRNA expression, morphological profiles, and ex vivo interactions with neurons. In Aim-2 we will determine if MB intervention (immediate or delayed) prevents or reverses glia-mediated inflammation and cognitive deterioration months after TBI. Cognitive ability will be assessed for 6 m after TBI. Parallel to these assessments, glial inflammatory states and associated axonal injury and myelination changes will be determined 1, 3, and 6 m after TBI. In Aim-3, we will determine if immediate MB intervention prevents TBI-induced immune-reactivity of microglia and the development of neuropsychiatric complications. To address this, mice will receive an immune challenge 1 m after TBI and glial profiles and depressive-like behavior will be determined. Collectively, completion of these aims will provide new insight into TBI-induced glial priming and immune-reactivity and will address the efficacy and long-term benefit of methylene blue intervention.